Benchtop Flow Cytometry Solutions - Kits & Reagents

TM

TM

TM

guava

Benchtop Flow Cytometry Kits and Reagents

http://www.millipore.com/?cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/flowcytometry/fc/fchome&cid=BIOS-S-EPDF-1031-1105-DS

WHAT’S INSIDE

Cell Health• Cell Counting & Viability• Cell Cycle• Mitochondrial Analysis• Apoptosis

Cell Signaling• MAPK Pathway• EGFR Pathway• PI3/Akt/mTOR Pathway• Jak/STAT Pathway• DNA Damage Pathway• Chemokine

Stem Cells• Embryonic Stem Cell

(Human/Mouse)• Neural Stem Cell (Rodent)

Immunology• Regulation T-Cell• Phenotype Markers

Milli-Mark™Conjugated Antibodies

Components of the guava® Flow Cytometry Solution• Instruments: easyCyte™

Flow Cytometers • Software• Kits & Reagents

FROM CORE LAB TO YOUR LAB—GUAVA® INTEGRATED FLOW CYTOMETRY SOLUTIONSFlow cytometry is an essential tool for in-depth cell analysis. With the capacity to simultaneously measure multiple parameters on hundreds of individual cells per second, flow cytometry offers greater speed, precision, and detail than most other cell analysis methods available to scientists today. Integrated products from Millipore will help streamline your workflow. Our complete benchtop flow cytometry solution includes our instruments, assays, and software—as well as the cell isolation and culture tools to prepare your samples. With a flow cytometry solution right in your lab, you’ll experience superior performance, higher quality data and faster progress from hypothesis to results.

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Antibody Selectionand Assay DesignThe antibodies used in each kit are carefully selected by reviewing many publications.

Ab Component ValidationAntibodies are screened primarily by Western blot to determine specificity, then validated with secondary antibody for flow cytometry use.

Ab Conjugation and TestingAntibodies are conju-gated to fluorophores that will ensure less fluorescence spectrum overlap.

Multiplexing, Stability, Performance ClaimsThe antibody conjugates are optimized to provide the best signal-to-noise ratio when multiplexing.

FlowCellect Kits and Milli-MarkConjugated AntibodiesMany researchers invest time optimizing and validating

antibodies for flow cytometry, only to discover that these

antibodies do not perform well when multiplexed together,

because of interference from the matrix or from other

antibodies.

Millipore’s FlowCellect kits and Milli-Mark conjugated

primary antibodies are fully optimized for fast, easy, and

accurate multiparametric flow cytometry. We’ve taken the

guesswork out of assay development so you can focus

on your results. We optimize and validate every antibody,

making sure they work together well. All you need are cells

and a research question; our assay kits will do the rest, and

you’ll have data before your cells are ready to split again.

FlowCellect Kits FlowCellect kits are Millipore’s proprietary multiparameter

flow cytometry kits for the analysis of cellular events

and/or cell phenotypes. Each kit has unique combination

of directly conjugated antibodies, and/or fluorescent

dyes and protein reporters to monitor changes in protein

expression and posttranslational modification. The kits also

contain complete buffer sets, protocols and pre-defined

gate settings. They are fully optimized for “plug-and-play”

cellular analysis on guava and other flow cytometers.

Advantages• Multiplexing capabilities

• Easy to use, with fewer incubation and wash steps

• Fully validated and concentration-optimized,

guaranteed to work in flow cytometry

Using a four-step validation process to develop our

FlowCellect kits, we’ve eliminated the need to design your

experiment or optimize antibodies and buffers:

Milli-Mark Conjugated Antibodies Milli-Mark antibodies are directly conjugated primary

antibodies that are validated for flow cytometry in

addition to traditional applications like Western blotting

and immunocytochemistry (see figure 1b for an example

of antibody validation). Because of their extensive cross-

platform validation, Milli-Mark antibodies are valuable,

convenient building blocks with which you can configure

your own assays.

FlowCellect Kit Development Steps

250

130

100

70

55

35

27

15

10

10

15

27

35

55

70

100

130

250

44kDa

OCT-4 antibody

SSEA-4 antibody

70kDa

Fig. 1. 1a. Mouse embryonic stem cells and fibroblasts labeled with Oct-4 (green), SSEA-1 (red), and Hoechst nuclear stain (blue). 1b. Human embryonic stem cell lysate.

Kits and Reagents


2

Knowing the performance profile of your cells prior to

running your bioassay can mean the difference between

valid assay results and wasted reagents, lost time and

discarded data.

Monitoring key indicators of cell health, such as

the apoptotic fraction and stage of apoptosis, viability,

cell cycle, cell counts, transfection efficiency, and target

expression levels, helps establish uniform standards of

cellular performance across long-term research studies.

These standards can be applied to a wide range of

bioassays. Whether you are establishing screen/no screen

criteria for high throughput screening, monitoring and

optimizing bioreactor conditions, or eliminating sources of

assay variability, consistent monitoring of your cell model

improves your bioassay performance and productivity.

CELL COUNTING AND VIABILITYCell counting and viability assessments can be used in a

variety of applications, such as cytotoxicity studies, PBMC

counting and rapid apoptosis assessment.

guava ViaCount® Assay KitThe guava ViaCount assay is fast becoming the new

standard for viability and cell counting. In this simple

no-wash, mix-and-read-assay, you can accurately obtain

absolute total cell counts, perform viability assessments

and determine apoptotic percentages—all from tiny

samples. You’ll enjoy several advantages over traditional

methods, including greater accuracy, reproducibility and

speed.

Cell Signaling• Chemokine

Viability Stain

Nuc

leat

ed C

ells

Live

Apoptotic

Dead

Annexin V

Via

bilit

y Sta

in

Live

Apoptotic

Dead

FL3

-Hei

ght

FL2-Height

2

FL2

-Hei

ght

2

Fig. 2. The blue population of cells show a significant amount of annexin V staining (right plot), indicating that intermediate levels of staining with the viability dye correlates with apoptosis.

ORDERING INFORMATIONDescription Qty/Pk Catalogue No.

Guava ViaCount Reagent Kit 100 Tests 4500-0040

Guava ViaCount Reagent Kit 600 Tests 4500-0041

Guava ViaCount Flex Reagent Kit for challenging samples

100 Tests 4500-0110

Guava ViaCount Flex Reagent Kit 500 Tests 4700-0060

Guava ViaCount Cell Dispersal Reagent

100 Tests 4700-0050

Cell Health

AdvantagesAssay:• Simple no-wash, mix-and-read procedure

• Counts up to 10 times faster than manual methods

• More reproducible than traditional testsSamples:• Uses small samples in tubes or a 96-well plate

• Handles low-density and small-volume cell samples

• Works with adherent or suspended cells and mammalian and insect cells

Discover the power of flow cytometry for multiparametric cell health analysis.


http://www.millipore.com/catalogue/item/4500-0040&open&cid=BIOS-S-EPDF-1031-1105-DS





3

Discover the power of flow cytometry for multiparametric cell cycle research.

CELL CYCLECell cycle phase distributions can be used to assess

cell health and proliferation and studying the potential

mechanism of antineoplastic agents. For example,

measuring the population of S phase cells can reflect the

amount of newly synthesized DNA. Also, distinguishing cells

in G2 from M phase cells can help identify cells undergoing

mitosis.

Flow cytometry analysis of cell DNA content has been

one of the best and most popular tools for researchers

to be widely used for the estimation of cell cycle phase

distribution. However, the limitation of single-marker

analysis, such as a DNA dye only, is that cells within

each phase cannot accurately be determined without

mathematical interpolation using analysis software.

The cell cycle can be divided into two distinct stages.

The first stage is interphase which consists of the G1, S,

and G2 phases, in which cells are active, growing, and DNA

is being replicated. The second is M-phase, also known as

the “mitotic phase”, in which cell division takes place.

FlowCellect Cell Cycle Kits Millipore has developed and optimized two bivariate cell

cycle analysis kits using phase-specific antibodies in

addition to a DNA dye. Bivariate analysis will not only reveal

the cell distribution within a particular phase of cell cycle,

but can also enable the researcher to elucidate mechanisms

of cell cycle regulation, without sophisticated software

modules or algorithms.

Cell Growth

Cell Prepares for Division

DNA Synthesis

Mitosis

Cells that cease dividing

I N T E R P H A S E

Cytokinesi s

FEATURED PRODUCT

FlowCellect Bivariate Cell Cycle Kit for DNA Replication Analysis Investigate DNA replication in the S phase with high

accuracy and confidence. The kit includes a directly

conjugated Anti-BrdU Alexa Fluor® 488 antibody plus

a DNA dye (propidium iodide). BrdU incorporation is a

widely accepted method of measuring DNA replication

and kinetics of cell cycle progression. The percentage of

BrdU labeled cells is a reliable estimate of the S phase

compartment, and labeled cells can then be followed

through the cell cycle.

Fig. 3. Detection of DNA replication by analysis of S phase cells. Bivariate flow cytometric analysis us-ing BrdU Alexa Fluor 488 conjugate can distinguish S phase cells with great accuracy, not only based on their difference in DNA content from G1 or G2/M cells but also as having incorporated BrdU.

G=24% (-BrdU, 1X DNA content)

S=72% (↑BrdU, 1-2X DNA content) G2/M=4% (-Brdu, 2X DNA content)

Advantages• No specific cell cycle analysis software required

• Quantitative measurements of percentage of cells

within each cell subpopulation

• Minimal assay development needed

• Includes all optimized flow cytometry antibodies

and buffers

4

FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis (Application)

Fig. 5. Nocodazole treatment increases percentage of cells in M phase. Cell were either treated with 100 µm Nocodazole (test sample) or left untreated (control) overnight at 37 ºC. By plotting the phosohorylation of histone H3 at Ser10 (y axis) versus DNA content (x axis), an increase in the proportion of G2/M cells was observed indicating that mitotic cells have accumu-lated after treatment. Apporomately 2% of cells reside in M phase under normal conditions in Jurkat cells, but when treated cell population increased 18%.

guava Cell Cycle AssayThis kit uses the nuclear DNA stain, propidum iodide (PI), to

measure cell cycle. Resting cells (G0/G1) contain two copies

of each chromosome. Cycling cells synthesize chromosomal

DNA (S phase), which results in increased fluorescence

intensity. When all chromosomal DNA has doubled (G2/M

phase), cells fluoresce with twice the intensity of the initial

population.

ORDERING INFORMATION FlowCellect Kits(using phase-specific antibodies plus a DNA dye)

Description Qty/Pk Catalogue No.

Bivariate Cell Cycle Kit for DNA Replication Analysis Anti-BrdU / Propidium Iodide Solution

25 tests FCCH025102

Bivariate Cell Cycle Kit for G2/M Analysis Anti-phospho-Histone H3(Ser10) / Propidium Iodide Solution

25 tests FCCH025103

FlowCellect Cell Cycle Check Point ATM DNA Damage Kit

25 tests FCCH025143

Flow Cellect Cell Cycle Check Point H2A.X DNA Damage Kit

25 tests FCCH025142

guava Cell Cycle Reagent Propidium Iodide Solution

100 tests 4500-0220

Cell Cycle Phases:G1 = 57%S = 19%G2 = 15%M = 3%

FEATURED PRODUCT

FlowCellect Bivariate Cell Cycle Kit for G2/M AnalysisInvestigate the G2/M phase transition with high

accuracy and confidence. The phosphorylation of

histone H3 at Ser10 correlates with the G2 to M

phase transition and is a prerequisite for chromatin

condensation at mitosis. At the end of mitosis,

histone H3 is rapidly dephosphorylated and remains

unphosphorylated throughout the remainder of

interphase. Therefore, phospho-histone H3 (Ser10) is

a reliable, specific marker of M-phase cells.

Fig. 4. Discrimination between G2 and M phase cells by measuring the phosphorylation of histone H3 on Ser10. Histone H3 is constitutively phosphorylated at Ser10 during metaphase.

http://www.millipore.com/catalogue/item/FCCH025102&open&cid=BIOS-S-EPDF-1031-1105-DS





5

Mitochondrial Signaling and ApoptosisExtrinsic pathway signal

Smac/DIABLO

OMI/HTRA2

Pro-Caspase 8Caspase 8

Caspase 8

Caspase 3Caspase 3

Casp 9

Apoptosome

ER stress,DNA damageOxidants

Pro-Caspase 9

APAF-1

EndoG

AIF

DNA fragmentationchromatin condensation

Bidt-Bid

t-Bid

Type II

Bax, Bak,

Type I

IAPIAP

Cyt c

Cyt c

Adapted from Bayir and Kagan Critical Care 2008 12:206.

MITOCHONDRIAL ANALYSISMitochondria are critical cellular organelles that produce

90% of cellular energy, control cell survival by regulating

apoptosis, and produce reactive oxygen species (ROS).

Mitochondrial superoxide generation results in of oxidative

stress, damage and cell death by apoptosis or to a cellular

energetic decline. Therefore, mitochondrial dysfunction

caused by disease or compound treatment has dire

consequences that can result in apoptosis, necrosis/cell

death, or caspase-independent cell death.

Monitoring impact on mitochondria and related cell

health markers is an important part of drug screening

programs, pathway mapping, and apoptosis and disease

research.

Flow cytometry detects multiple markers simultaneously

at various stages of apoptosis, making it a powerful

technique for studying pathways governing cell health and

cell death.

Discover the power of flow cytometry for multiparametric mitochondrial analysis.

FlowCellect Mitochondrial Kits These kits harness the power of flow cytometry to assess

changes in mitochondrial membrane potential, apoptosis

as measured by Annexin V binding, mitochondrial oxidative

stress, and cell death, using only minimal cellular samples.

The kits may be used with most dual laser flow cytometry

systems equipped with a 488 nm and a 644 nm laser.

AdvantagesAssay:• Multiplex detection with no optimization required

• Highly reproducible


• All optimized flow cytometry antibodies and buffers

included

• Enables novice users to perform complex analysis

Samples:

• Designed to run 100 samples of human cells

FlowCellect MitoDamage KitKit Component Laser Buffer Pack

MitoSense Red Dye Red

10X Assay Buffer HSCAnnexin CF 488 Blue

7-AAD Blue

Simultaneously measures 3 important cell health

parameters:

• Change in mitochondrial potential (considered an early

hallmark of apoptosis or cell stress)

• Phospatidylserine translocation to the surface of early

apoptotic cells (measured by Annexin V binding)

• Plasma membrane permeabilization or cell death

(measured by 7-AAD)

Fig.6. Dot plots depicting Jurkat cells stained using the MitoDamage kit. Jurkat cells uninduced (column A), induced to apoptosis with 2 µM staurosporine (column B) or with 50 µM CCCP (column C), then stained using the MitoDamage kit. Plots show the percentage of positive cells for:

1st row: Apoptosis (Annexin V binding) and mitochondrial membrane potential change 2nd row: Cell death and mitochondrial membrane potential change 3rd row: Apoptosis and cell death. Data reports that 2 µM staurosporine induces apoptosis in Jurkat cells, and that 50 µM CCCP depolarizes the mitochondrial

membrane, but neither condition is sufficient for cell membrane permeabilization and death.

The FlowCellect MitoDamage kit can thus distinguish

multiple populations:

1. Healthy cells with intact mitochondrial membrane

2. Stressed cells with dissipated membrane potential

without Annexin V or 7-AAD staining

3. Early apoptotic cells with dissipated membrane

potential and Annexin V binding

4. Late apoptotic cells or dead cells with dissipated

membrane potentials

Mit

oSen

se R

ed

Annexin V, CF488A

Mit

oSen

se R

ed7-A

AD

Annexin V, CF488A

Red

2 F

luor

esec

ence

(R

D2

-HLo

g)

Green Fluorescence (GRN-HLog)

94.4%

0.75% 3.7%

Uninduced

100 101 102 103 104100

101

102

103

104

1.1%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

95.2%

3.2% 1.3%100 101 102 103 104

100

101

102

103

104

0.3%

Red

2 F

luor

esce

nce

(RD

2-H

Log)


0.16%

95.2% 3.2%100 101 102 103 104

100

101

102

103

104

1.4%

Red

2 F

luor

esce

nce

(RD

2-H

Log)


0.06%

70.5% 28.2%100 101 102 103 104

100

101

102

103

104

1.2%

Red

2 F

luor

esce

nce

(RD

2-H

Log)


0.10%

93.9% 4.8%100 101 102 103 104

100

101

102

103

104

1.2%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluorescence (RED-HLog)

0.26%

98.4% 1.3%100 101 102 103 104

100

101

102

103

104

0.02%

Red

2 F

luor

esec

ence

(R

D2

-HLo

g)


54.7%

14.6% 27.0%100 101 102 103 104

100

101

102

103

104

3.7%

Red

2 F

luor

esec

ence

(R

D2

-HLo

g)


0.20%

93.2% 6.6%

50 µM CCCP

100 101 102 103 104100

101

102

103

104

0.04%

100 101 102 103 104

101

102

103

104

Depolarized Cells

Dead Cells

Live Cells

58.1% 0.08%

41.0% 0.8%

Red

2 F

luor

esce

nce

(RD

2-H

Log)

Red Fluoresecence (RED-HLog)

100

2 µM Staurosporine

7-AAD

FEATURED PRODUCT

6


7

FlowCellect MitoPotential Red KitSimultaneous analysis of mitochondrial membrane potential

along with cell death provides key information for drug

discovery, cancer and toxicology studies as well as disease-

induced apoptosis. This kit uses MitoSense Red (a red,

laser-excitable dye) to monitor mitochondrial membrane

potential changes in early apoptosis, and 7-AAD (a live-cell-

impermeant DNA intercalator) to simultaneously monitor

cell membrane permeability changes in late apoptosis and

necrotic cell death.

FlowCellect MitoLive KitIncludes:

• MitoSense Red, a fluorescent cationic dye that

accumulates in the mitochondria and is responsive to

mitochondrial potential changes

• Calcein acetoxymethylester (calcein-AM) a non-

fluorescent, cell-permeant compound that is hydrolyzed

by intracellular esterases into the fluorescent anion

calcein and provides a measure of cellular vitality.

The simultaneous use of the reagents enables researchers

to obtain information on early and late apoptosis in one

simple assay.

FlowCellect MitoStress KitIncludes:

• MitoSOX™ Red, a live-cell-permeant, fluorogenic dye which

targets the mitochondria and reacts with superoxide

radicals and fluoresces yellow/red

• Annexin V conjugated to CF647 which binds to

phosphatidylserine (PS) on the surface of apoptotic cells

The simultaneous use of these reagents enables

researchers to obtain information on oxidative stress and

apoptosis in one simple assay.

FlowCellect Cytochrome c KitIncludes a directly labeled anti-Cytocrome c–FITC antibody

and Anti-IgG1-FITC isotype control. Viable or live cells will

demonstrate higher levels of Cytochrome c staining while

apoptotic cells which have released their Cytochrome c

from the mitochondria to the cytoplasm will demonstrate

reduced staining intensity. The FlowCellect Cytochrome c

flow cytometry kit is a simple, gentle, and fast method to

assess levels of Cytochrome c in mitochondria, providing a

valuable tool for assessing proapoptotic signaling and the

efficacy of proapoptotic anti-cancer agents in cells.

ORDERING INFORMATION FlowCellect Mitochondrial Kits

Description Qty/PkCatalogue No.

FlowCellect MitoPotential Red Kit • Two dyes for measuring cell death

and mitochondrial membrane potential

• MitoSense Red (Red Laser) / 7-AAD (Blue Laser)

100 tests FCCH100105

FlowCellect MitoDamage Kit • Three dyes to assess

mitochondrial potential, stress, and cell death

• Mitosense Red (Red) / Annexin CF 488 (Blue) / 7-AAD (Blue)


FlowCellect MitoLive Kit • Two dyes to measure

mitochondrial health and cell vitality

• Mitosense Red (Red) / Calcein-AM (Blue)


FlowCellect MitoStress Kit • Understanding the regulation of

apoptosis and oxidative stress.• MitoSox Red (Red) / Annexin V 647

(Blue)


FlowCellect Cytochrome c Kit• Easy way to detect loss of

mitochondrial Cytochrome c in cells

• Anti-Cytochrome c (Blue) / IgG1 (Blue)


FlowCellect Oxidative Stress Characterization Kit• Detection of oxidative stress by

flow cytometry• Anti-DNP (Blue)

25 tests FCCH025111

guava Mitochondrial Depolarization Assay Kit• Monitoring changes in

mitochondrial membrane potential• JC-1 (Blue) / 7-AAD(Blue)

100 tests 4500-0250












APOPTOSISCells respond to specific apoptotic signals by initiating

intracellular processes that result in characteristic

physiological changes. Among these changes are

externalizations of phosphatidylserine to the cell surface,

depolarization of mitochondrial membranes, cleavage

and degradation of specific cellular proteins, compaction

and fragmentation of nuclear chromatin, loss of cell

membrane integrity, and cellular shrinkage. Suppression or

enhancement of apoptosis is known to cause or contribute

to diseases such as cancer and diabetes, making the

apoptotic pathway a popular drug target.

Because apoptosis is a dynamic event, and the time

period during which cells exhibit apoptosis markers is

variable and short, flow cytometry is an ideal technique

for tracking cells through apoptosis. Our easy-to-use kits

enable you to examine cells at each of the various stages

of apoptosis.

Early Apoptosis Flow Cytometry Kits Two separate dyes identify a broad spectrum of

apoptotic and non-apoptotic cells: Annexin V binds to

phosphatidylserine on the external membrane of apoptotic

cells, while 7-AAD permeates and stains DNA of late-stage

apoptotic and dead cells.

Mid-Apoptosis Flow Cytometry Kits Caspase activity is measured using a FLICA (fluorescent

labeled inhibitor of caspase) reagent, supplemented by

a nuclear DNA stain 7-AAD, which evaluates membrane

integrity and cell viability. The assays are available in two

forms, sulforhodamine (SR) and carboxyfluorescein (FAM),

giving greater flexibility in assay design as well as the

capacity to multiplex caspase assays.

Late Apoptosis Flow Cytometry Kits The guava TUNEL assay detects apoptosis-induced DNA

fragmentation through a quantitative fluorescence assay.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the

incorporation of bromo-deoxyuridine (BrdU) residues into

the fragmented nuclear DNA at the 3’-hydroxyl ends. A

TRITC-conjugated anti-BrdU antibody then labels these

DNA fragments. The assay distinguishes two populations:

non-apoptotic cells (TUNEL-negative) and apoptotic cells

(TUNEL-positive).

Live/Healthy(non-committed)

cells

FLICA(-)PI(-) FLICA(+)PI(-) FLICA(+)PI(+) FLICA(-)PI(+)

Early/Mid-stage(committed) apoptotic

cells

Late stageapoptotic/dying

cells

Dead cells

C+C+C+

C+

C+C+ C+

C+PI+PI+

PI+PI+

PI+PI+


cells



cells


cells

Dead cells

C+C+C+

C+

C+C+ C+

C+PI+PI+

PI+PI+

PI+PI+


cells



cells


cells

Dead cells

C+C+C+

C+

C+C+ C+

C+PI+PI+

PI+PI+

PI+PI+


cells



cells


cells

Dead cells

C+C+C+

C+

C+C+ C+

C+PI+PI+

PI+PI+

PI+PI+

Advantages• Two dye strategy : Detect various stages of apoptosis

within a one assay

• Mix-and-read assay: Get standardized results even with

multiple users

• All-in-one-kit : Spend less time before analysis

• Compatible with pairing with FITC or PE probes or other

probes in the green or yellow channels.

• Probe with other markers in green and yellow channels

with the FlowCellect Mitochondrial kits.

Early Mid LateGuava Nexin® Multicaspase Guava TUNELAnnexin Red Caspase 3/7, 8, 9 Assay

MitoPotential Red Dual CaspaseMitoDamageMitoStressMitoLiveCytochrome c

c

cc

c

c+

PS

PS

PS

PS

PS

PSc+

c+

c+

c+

c+

G...CG...C

C...GA...T

G...CC...G

A...T

A

GG...C

G...C

C...GA...T

G...CC...G

A...T

A

GG...C

G...C

C...GA...T

G...CC...G

A...T

A

G

TdT+

BrdUTP

DNA strand breaks due to

apoptosis

Add BrdUTPto 3’OH ends

Antibody labeledbreak sites

TRITC-Anti-BrdU

How the guava TUNEL Assay Works

Discover the power of flow cytometry for multiparametric apoptosis analysis.


cells

Early/Mid-stage(committed)

apoptotic cells


cells

Dead cells


DNA strandbreaks due to apoptosis

Add BrdUTPto 3’OH ends

Antibody labeledbreak sites

8

9

FlowCellect Annexin Red KitTwo Dyes to distinguish early Apoptosis from later stages

PS PSPS

PS

PS = phosphatidylserine

Healthy Cell Membranes

PSPSPSPS

Apoptotic Cell Membranes

PSPS

PSPS

PS

PS= 7AAD

Annexin CF647

PS PSPS

PS

PS = phosphatidylserine

Healthy Cell Membranes

PSPSPSPS

Apoptotic Cell Membranes

PSPS

PSPS

PS

PS= 7AAD

Annexin CF647

In the early stages of apoptosis, phosphatidylserine

molecules, which can bind to Annexin V, move from

the inner leaflet, to the outer leaflet of the plasma

membrane. A rapid, sensitive, and convenient assay to

monitor early and late apoptosis, the FlowCellect annexin

red kit includes recombinant Annexin V conjugated to

the red sensitive dye CF647, and 7-AAD (a live cell-

impermeant dye) to measure cell membrane integrity.

After staining cells with FlowCellect Annexin Red kit,

three populations of cells can be identified in this assay:

• Non-apoptotic cells: Annexin V(-) and 7-AAD(-)

• Early apoptotic cells: Annexin V(+) and 7-AAD(-)

• Late-apoptotic or dead cells: Annexin V (+) and

7-AAD(+)

Kit Description Laser Buffer Pack

Annexin V, CF 647 Red 10X Assay Buffer

7-AAD Blue

Fig. 7. Dot plots depicting Jurkat cells stained using the FlowCellect Annexin Red kit. Jurkat cells were untreated (Plot A), treated with 0.1 µM (Plot B) or with 3 µM staurosporine (Plot C), and then stained using the FlowCelllect Annexin Red kit. The percentage of apoptotic cells increased from 36% to 92% in response to a 30-fold increase in staurosporine concentration; however, only a small fraction (< 10%) of the cells showed evidence of cell death.

7-A

AD

B. C.

Annexin V

A.

Red

2 F

luor

esce

nce

(RD

2-H

Log)

2% 2%

85% 11%

Red2 Fluorescence (RED2-HLog)

100 101 102 103 104100

101

102

103

104

100

101

102

103

104

Red

2 F

luor

esce

nce

(RD

2-H

Log)

4%

60% 36%


100 101 102 103 104100

101

102

103

104

Red

2 F

luor

esce

nce

(RD

2-H

Log)

7%

5% 92%


100 101 102 103 104

3 µM Staurosporine0.1 µM StaurosporineUninduced

ORDERING INFORMATION Apoptosis Kits Description Qty/Pk Catalogue No.

Early Apoptosis Kits

FlowCellect Annexin Red Kit (Annexin V-CF647) 100 tests FCCH100108

guava Nexin Kit (Annexin V-PE) 100 tests 4500-0450

Mid-Apoptosis Kits (For a complete listing visit: millipore.com/midapoptosis_kits)

guava MultiCaspase SR Kit 100 tests 4500-0500

guava MultiCaspase FAM Kit 100 tests 4500-0530

guava MultiCaspase 3/7 FAM Kit 100 tests 4500-0540

guava MultiCaspase 3/7 SR Kit 100 tests 4500-0510

Late Apoptosis Kit

guava TUNEL Reagent Kit 100 tests 4500-0121

FEATURED PRODUCT




http://www.millipore.com/flowcytometry/fc4/apoptosis_cancer&open&cid=BIOS-S-EPDF-1031-1105-DS






Signal transduction pathways lead to diverse outcomes,

such as apoptosis, cell differentiation, cell growth and cell

proliferation, all of which have been extensively studied in

the process of developing therapies for various cancers

and autoimmune disease. Cross-talk among signaling

pathways adds an extra dimension of complexity when

analyzing physiological consequences of a pathway of

interest. However, there are some key nodes at which

multiple signals are integrated. Multiparametric flow

cytometry provides researchers the power to monitor

these intracellular ‘checkpoints’ simultaneously, enabling

analysis of complicated cell events.

FlowCellect Cell Signaling Kits The study of cell signaling has been made easier by

activation status-specific and phospho-specific antibodies.

Measuring the activity of cell signaling pathways by flow

cytometry delivers robust, high content information in

less time than traditional methods by analyzing multiple

parameters on hundreds of cells per second.

Dual Detection FlowCellect Kits With Pairs of Total and Phospho-Specific Antibodies

We carefully selected pairs of antibodies that bind to

the same protein target; one to detect total protein

expression and another to detect the phosphorylated

from of the same protein. By using two-color

analysis we can achieve target-specific detection

of phosphorylation and, by doing so, eliminate false

positives in mixed cell populations. Performing dual

detection of treated cells reveals an accurate,

biologically relevant activation state for a given protein.

Implementing this technique can prove to be a powerful

tool in assessing small molecule activity and specificity

in drug screening campaigns.

AdvantagesAssay:• Ensures specific labeling of targets

• Multiplex detection with no optimization required


Samples:• Designed to run 25 samples of human cells

EGFR and MAP Kinase Kits• MAPK Activation Dual Detection Kit - Includes two

directly conjugated antibodies, anti-phospho-ERK1/2

(Thr202/Tyr204, Thr185/Tyr187)-PE and anti-ERK1/2-

Alexa Fluor 647 antibody to measure total levels of

ERK. This two-color flow cytometry kit is designed to

measure the extent of MAPK phosphorylation relative to

the total MAPK expression in any given cell population.

• PI3K/MAPK Dual Pathway Activation and Cancer Marker

Detection Kit

• EGFR/MAPK Pathway Activation Detection Kit

• EGFR RTK Activation Dual Detection Kit

PI3K / Akt / m-TOR Kits:• FlowCellect PI3K-mTOR Analysis Kit - This kit includes

two directly conjugated phospho-specific signaling

antibodies which are optimized for multicolor flow

cytometry to analyze the mTOR pathway in great

detail. The phosphorylation of Akt is indicative of the

upstream PI3K signaling, marking the cell’s initiation into

proliferation or cell survival. Phosporylated ribosomal S6

protein is indicative of downstream mTOR and p70S6K

signaling leading to protein translation.

• PI3K Activation Dual Detection Kit

Cell Signaling

Discover the power of flow cytometry for multiparametric cell signaling research.

FlowCellect Cell Signaling Kits With Directly Conjugated, Phospho-Specific Antibodies

Determine the effect of mechanical and chemical

reagents that can induce DNA damage, discern multiple

pathway activation and cross talk in a time-dependent

manner, or study the correlation between pathway

activation and changes in cell function and health.

10


11

FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection KitThree antibodies to study cross talk between the PI3K and

MAPK pathways. The kit uses directly labeled antibodies

against phospho-Akt1/PKBα(Ser473) and phospho-Erk1/2

(Thr185/Tyr187) to analyze signaling activation and cross

talk, plus Ki-67 marker to identify the proliferative fraction.

Together, the antibody trio makes it easy to evaluate

the role these signaling pathways play in proliferation

and differentiation. The kit also includes Cell Cycle Stop™

fixation reagent to improve Ki-67 detection. Although Ki-67

is present throughout most of the cell cycle, it is difficult

to detect by flow cytometry except during M phase. Cell

Cycle Stop reagent arrests the cycle at the M phase,

making it possible to accurately detect Ki-67 expression

and discern the biological effects of the PI3K/MAPK cross

talk.

Kit Description Laser Buffer Pack

Anti-Phospho-Erk1/2 (Thr202/try204, Thr185/Tyr187)

Alexa Fluor 488

Cell Cycle Stop Fixation Buffer Wash Buffer Assay Buffer Permeabili-zation Buffer

Anti-Phospho-Ark1/PKBα (Ser473)

R-Phycoerythrin

Anti-Ki-67 PerCP

Fig. 8. HEK293 cells were stimulated by insulin (B, E) or PMA (C, F) for three or five minutes, and simultaneously stained with antibodies against phospho-Akt and phospho-ERK1/2. The cross-talk between the PI3K/MAPK signaling pathways is demonstrated by the sharp decrease in ERK phosphorylation after five minutes of insulin stimulation.

1040.97% 0.47%

0.30%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

98.26%

1040.78% 61.94%

30.37%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

6.91%

104 100%90%80%70%60%50%40%30%20%10%

0%pAkt pErk

Untreated100 nM Insulin 3 min100 µg/mL PMA 3 min

Ki67

30.30% 60.06%

1.28%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

8.35%

104 1.04% 0.31%

0.18%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

A. Untreated B. Insulin Treated C. PMA Treated

D. Untreated E. Insulin Treated F. PMA Treated

HEK293

HEK293

3-M

inut

e Sti

mul

atio

n

3-Minute Stimulation

5-M

inut

e Sti

mul

atio

n

5-Minute Stimulation

pER

K-P

E

98.47%

104 0.72% 15.46%

75.15%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

8.67%

104 34.29% 49.52%

3.46%

103

102

101

100

100 101 102 103 104

pAKT-Alexa 488

pER

K-P

E

12.73%

100%90%80%70%60%50%40%30%20%10%

0%pAkt pErk Ki67

GTP

GDP

Shc

Grb2

Ki 67

PI3-Kinase

Proliferation/Survival

A CancerProliferative Marker

Growth/Differentiation

Wortmannin or LY294002

PMA

p85

IGF-1

p110 RAS

Raf-1Akt

P

P

P

P

P

SOS

MEK1/2

Inhibiting

Activating

ERK1/2

P

FEATURED PRODUCT


Fig. 9. Dual Parameter Analysis of Total and Phospho ERK 1/2 on Jurkat Cells. Analysis of total (x axis) and phospho (y axis) ERK on stimulated Jurkat cells stained with both anti-pERK1/2-PE and anti-ERK-Alexa Fluor 647 confirms target specificity of the phosphorylation event as indicated by the double positive cell population (9b). Stimulated Jurkat cells stained only with isotype control antibody showed no double positive cells (9a).

FlowCellect MAPK Activation Dual Detection Kit

Jak/STAT Kits• FlowCellect Multi-STAT Activation Kit – This kit is

designed to simultaneously detect the phosphorylation

of the most commonly studied STAT proteins through

cytokine or growth factor-mediated pathway activation.

The kit also enables a researcher to quickly profile

the status of constitutive activation of STAT1, STAT3,

and STAT5 within a population of cells or tumors. The

ability to detect the activation of multiple STAT proteins

simultaneously enables profiling of STAT signaling

pathways within a cell type of interest. Alterations in

these signaling profiles can be used to monitor the

effect of a particular upstream treatment targeting the

STAT pathways.

• EGFR/STAT3 Pathway Activation Detection Kit

DNA Damage Kit:Millipore’s FlowCellect multicolor DNA damage response

kit enables fast and easy detection of the phosphorylation

state of ATM, SMC1 and histone H2A.X by flow cytometry.

Millipore’s FlowCellect multicolor DNA damage response kit

was developed and tested using the DNA damaging reagent,

etoposide, in HeLa cells, but the kit can be used with other

human cell lines to determine the effect of mechanical and

chemical reagents that can induce DNA damage through the

ATM- dependent pathway.

12

FEATURED PRODUCT

FlowCellect MAPK Activation Dual Detection KitMillipore’s FlowCellect MAPK activation dual detection

kit includes two directly conjugated antibodies, a

phospho-specific Anti-phospho-ERK1/2 (Thr202/Tyr204,

Thr185/Tyr187)-PE and an Anti-ERK1/2-Alexa Fluor 647

conjugated antibody to measure total levels of ERK. This

two-color flow cytometry kit is designed to measure the

extent of MAPK phosphorylation relative to the total

MAPK expression in any given cell. The levels of both

the total and phosphorylated protein can be measured

simultaneously in the same cell, resulting in a normalized

and accurate measurement of MAPK activation after

stimulation. Moreover, simultaneous measurement of both

total and phospho-ERK1/2 confirms target specificity of

the phosphorylation event. Together, a total and phospho

antibody duo used in multiplex provides an enhanced and

more reliable detection of the phosphor: total ratio within

a mixed population.

9a.

9b.

http://www.millipore.com/catalogue/item/FCCS025106&open&cid=BIOS-S-EPDF-1031-1105-DS


13

ORDERING INFORMATION Chemokine Receptor Kits Description Qty/Pk Catalogue No.

Flowcellect Chemokine Receptor CCR2B Surface Expression Quantification Kit

100 tests

FCCR2004411

Flowcellect Chemokine Receptor CCR4 Surface Expression Quantification Kit

100 tests

FCCR4004413

Chemokine Receptor CCR6 Surface Expression Quantification Kit

100 tests

FCCR604414

Flowcellect Chemokine Receptor CCR7 Surface Expression Quantification Kit

100 tests

FCCR7004415

AdvantagesAssay:• Includes pharmacologically characterized positive

and negative control cells

• Same accuracy as radioactive assays, without the

hazards.

• Ability to identify high, medium, and low expressing

cell cultures during the clonal selection process.

• High reproducible results obtained by using

Millipore’s well-characterized ChemiScreen™ GPCR

cell lines as assay controls

Samples:• Sufficient reagents to run 100 samples of human

cells.

FlowCellect Chemokine Receptor Surface Expression Quantification KitsMillipore offers 11 GPCR surface identification flow

cytometry kits. Flow cytometry provides high quality,

reproducible data in far less time than traditional methods

for chemokine receptor research and avoids the hazard and

expense of radioligand binding assays.

Our FlowCellect GPCR identification kits can be used to

identify and quantify GPCRs on the surface of any cells.

The kits detect GPCR expression using a GPCR-specific

antibody validated for flow cytometry. Also included are

positive and negative control cells with well-characterized

receptor expression levels for the purposes of quantitative

extrapolation.

For a complete listing of kits visit: millipore.com/flowcytometry

ORDERING INFORMATION Cell Signaling Kits Description Qty/Pk Catalogue No.

PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit• Anti-phospho-Erk1/2 (Thr202/Tyr204,

Thr185/Thr187)-R-Phycoerythin• Anti-phospho-Akt1/PKBα

(Ser473) – Alexa Fluor 488• Anti-Ki-67-PerCP

25 tests FCCH025100

Multicolor DNA Damage Response Kit• Anti-phospho-SMC1

(S957)- Alexa Fluor 488• Anti-phospho-ATM

(S1981)- PE• Anti-phospho-HistoneH2A.X

(S139)- PerCP

25 tests FCCH025104

EGFR/MAPK Pathway Activation Detection Kit• Anti-phospho-EGFR

(Tyr1173) – Alexa Fluor 488• Anti-phospho-Erk1/2 (Thr202/Tyr204,

Thr185/Thr187)-R-Phycoerythin

25 tests FCCS025101

PI3K Activation Dual Detection Kit• Anti-phospho-Akt

(Ser473)- Alexa Fluor 488• Anti-Akt/PKB-Alexa Fluor 647

25 tests FCCS025105

MAPK Activation Dual Detection Kit• Anti-phospho-Erk1/2

(Thr202/Tyr204, Thr185/Thr187)- PE• Anti-Erk1/2 –Alexa Fluor 647

25 tests FCCS025106

FlowCellect Bcl-2 Activation Dual Detection Kit

25 tests FCCSO25108

EGFR/STAT3 Pathway Activation Detection Kit • Anti-phospho-EGFR (Tyr1173)• Anti-phospho-pSTAT3 (Try705)

25 tests FCCS025111

FlowCellect p38 Stress Pathway Activation Detection Kit

25 tests FCCSO25132

PI3K-mTOR Signaling Cascade Mapping Kit• Anti-phospho-Ribosomal Protein S6

(Ser235) – PerCP• Anti-phospho-Akt1/PKBα

(Ser473) – Alexa Fluor 488

25 tests FCCS025210

Multi-STAT Activation Profiling Kit• Anti-phospho-STAT1

(Y701)- PerCP• Anti-phospho-STAT3

(Y705)- Alexa 488• Anti-phospho-STAT5A/5B

(Y694/Y699)- PE

25 tests FCCS025550

EGFR RTK Activation Dual Detection Kit• Anti-phospho-EGFR

(Tyr1173)- Alexa Fluor 488• Anti-EGFR-PerCP

25 tests FCCS025107












http://www.millipore.com/catalogue/item/FCCR2004411&open&cid=BIOS-S-EPDF-1031-1105-DS





Because flow cytometry has the power to characterize

subpopulations of cells within heterogenous cell mixtures;

it is widely used for studying both embryonic stem cells

(ESCs) and induced pluripotent stem (iPS) cells. Flow

cytometry enables researchers to evaluate percentages

of cells expressing specific markers, to determine culture

quality, and to track gene expression changes during a

differentiation protocol.

Embryonic Stem Cells (ESC)

Stem cell

Committed cell

Differentiated cells

(hESC, mESC)

(ENStem-A)Oct-4+

SSEA-4+ (human)TRA1-60+

HESCA+

Nanog+

SSEA-1+ (mouse)

Oct-4-/+

SSEA-4-

TRA1-60-

HESCA-

Oct-4-

SSEA-4-

TRA1-60-

HESCA-

SSEA-1+/-

FlowCellect Stem Cell Characterization KitsMillipore’s FlowCellect stem cell characterization kits

are designed to provide rapid, sensitive assessments of

embryonic and neural stem cell phenotypes at various

stages. The kits use three parameters for accurate

identification, enabling the research to “triangulate” cellular

phenotypes with two complementary positive markers and

one negative marker. The negative antibody also serves as

a stage- and species-specific or lineage-specific marker for

differentiated cells.

ORDERING INFORMATION Stem Cell Kits Description Qty/Pk Catalogue No.

Mouse Embryonic Stem Cell Nuclear Marker Characterization Kit• OCT-4, SSEA-1, SSEA-4

25 tests

FCMEC25110

Human Embryonic Stem Cell Nuclear Marker Characterization Kit• OCT-4, SSEA-4, SSEA-1

25 tests

FCHEC25102

Human Embryonic Stem Cell Surface Marker Characterization Kit• Tra-1-60, SSEA-4, SSEA-1

25 tests

FCHEC25106

Human Embryonic Stem Cell Surface Marker Characterization Kit• HESCA-1, SSEA-4, SSEA-1

25 tests

FCHEC25104

Rodent Neural Stem Cell Characterization Kit(Neural Differentiation)• SOX-2, Nestin, β-III-Tubulin

25 tests

FCRNC25112

Rodent Neural Stem Cell Characterization Kit(Astrocyte Differentiation)• SOX-2, Nestin, GFAP

25 tests

FCRNC25114

AdvantagesAssay:• All optimized, fluorescently-labeled flow cytometry

antibodies and buffers included

• Highly reproducible results

• Validated for flow cytometry, immunocytochemistry,

and Western blotting

Stem CellsDiscover the power of flow cytometry for multiparametric stem cell analysis.

14

http://www.millipore.com/stemcell/stm4/stemcellhome&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/catalogue/item/FCMEC25110&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/catalogue/item/FCHEC25102&open&cid=BIOS-S-EPDF-1031-1105-DS



http://www.millipore.com/catalogue/item/FCRNC25112&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/catalogue/item/FCRNC25114&open&cid=BIOS-S-EPDF-1031-1105-DS

15

FlowCellect Human Embryonic Stem Cell Characterization Kit The kit is provides a quick, easy way to track surface marker expression of hOCT4, SSEA4 and SSEA1. The

percentage of undifferentiated human embryonic stem cells in culture is reflected in the percentage cells

that express both hOCT4 and SSEA4 but not SSEA1. This quick test will enable researchers to determine the

multipotency of their cells in culture as well as see changes in marker expression during a differentiation protocol.

FlowCellect Human Embryonic Stem Cell HESCA-1 Surface Marker Characterization KitThis kit provides an easier and quick way to track surface marker expression of HESCA-1, SSEA4 and SSEA1.

The kit will also help to determine the percentage of undifferentiated human embryonic stem cells in culture by

determining the percentage of cells that express both HESCA-1 and SSEA4 and not SSEA1. This quick test will

enable researchers to test the quality of their cells in culture as well as see changes in marker expression during a

differentiation protocol.

Fig. 10. Representative data of H1 human embryonic stem cells stained with hOCT4-Alexa 488, SSFA4-PE and SSFA1-PF/CY5

10e40%

4.9%

0.1%

95%

10e3

10e2

10e1

10e010e0 10e1 10e2 10e3 10e4

SSEA

1-PE

/CY5

SSEA4-PE

A.

10e485.5%

14.4%

0.1%

0%

10e3

10e2

10e1

10e010e0 10e1 10e2 10e3 10e4

hOCT

4-Al

exa

488

SSEA1-PE/CY5

B. 10e40.1%

4.8%

85.5%

9.6%

10e3

10e2

10e1

10e010e0 10e1 10e2 10e3 10e4

hOCT

4-Al

exa

488

SSEA4-PE

C.A.

Fig. 11. Representative data of H1 human embryonic stem cells stained with HESCA-1-FITC, SSEA4-PE and SSEA1-PE/CY5.

B. C.A.

FEATURED PRODUCTS



The immune system, which mediates the body’s response

to the introduction of foreign material, is made up

of multiple cell types collectively called lymphocytes.

Lymphocyte subtypes include B cells (which secrete

antibodies), cytotoxic T cells, helper T cells (which secrete

cytokines), and natural killer (NK) cells. Characterization of

lymphocyte subtypes and cytokine signaling is essential for

understanding the complex nature of the immune system.

Activation by antigens, suppression of normal immune

activation, and disease states can affect the phenotypes

of lymphocytes. Multiparameteric phenotypic analysis

by flow cytometry allows researchers to distinguish one

subpopulation of cells within a heterogeneous mixture,

and thereby enables the study the dynamics of immune

signaling in intact cells.

FlowCellect Immunology KitsEach kit includes multiple optimized fluorescent labeled

antibodies and all buffers necessary for cell preparation

and analysis. Detailed assay instructions are included

to assist in analysis and to ensure that the correct cell

concentration is obtained during acquisition of sample data.

Fig. 12. Schematic diagram of CD4+ T-cell differentiation. Five different types of CD4+ T-cells can develop from a common naïve precursor depending on the cytokine environment and interaction with dendritic cells (DC).

AdvantagesAssay:• Multiplex detection with no optimization required

• Highly reproducible


• All optimized flow cytometry antibodies and buffers

included


Samples:• Designed to run 25 tests (FlowCellect kits)

• Designed to run 100 tests (guava kit)

DC

IL-12RTH1T-betSTAT4STAT1

IL-2RTREG

Foxp3STAT5

IL-21RTFHTHBcl-6

STAT3

IL-4RTH2GATA3STAT6

STAT5a

IL-23RTH17

RORγtSTAT3

IL-12

CD80

CD28

TCR

CD28

CD85

pMHCII

IFNγIL-18

IL-2IL-4IL-33

IL-6

IFNγ

IL-2

LTα

IL-21

IL-6TGFβ

IL-23

TGFβIL-2

IL-4

IL-5

IL-13

IL-25

IL-17

IL-17F

IL-22

IL-21

IL-21

IL-17

TGFβ

IL-10

IL-35

B ZONE

CD4+ T-Cell Differentiation

CD62Llo CCR7lo

CCR7lo

High TCR Binding

Low TCR Binding

Medium TCR Binding

CXCR5hi

CD62Lhi CCR7hi T ZONE

EMIGRANTnaive

ImmunologyDiscover the power of flow cytometry for multiparametric analysis of the immune system.

16

http://www.millipore.com/flowcytometry/fc4/immunology&open&cid=BIOS-S-EPDF-1031-1105-DS

FlowCellect Human Lymphocyte ZAP-70 Characterization KitIncludes three directly conjugated antibodies: CD5 FITC,

CD19 APC, and ZAP-70 PE to provide researchers the ability

to phenotypically distinguish cell types. CD5 is normally a

pan-T-cell marker, while CD19 is a mature B-cell marker.

Some B-cell malignancies express both of these molecules

on their surface, and therefore can be distinguished from

the two aforementioned cell types. ZAP-70 is a molecule

involved in T-cell activation, which can also be expressed

within B-cells at levels correlating to disease severity.

The combinatorial staining of cells for all three of these

markers assists the researcher studying the molecular

biology of T-cell activation or B-cell disease states.

Fig. 13. Mixture of Roamos (B cell line) and Jurkat (T cell Line) cells were used as a model system. The cells were then stained with CD5-FITC, CD19-APC, and ZAP-70PE. By drawing two gates on the dot plot showing CD5 vs. CD19, the expression levels of each of the three mark-ers were determined for the two cell subpopulations.

FlowCellect Mouse FoxP3 Treg Identification KitThis kit provides a quick and easy way to detect FoxP3

expression in mouse CD4+ T-cells. T regulatory (Treg) cells

represent 2 - 10% of the total peripheral CD4+ T-cells and

are characterized by the expression of the forkhead box

transcription factor, FoxP3. FoxP3 is a master regulator of

Treg development and function. Treg cells develop from

a common naïve CD4+ T-cell precursor and expression of

FoxP3 is induced in the presence of TGFβ and IL-2. Lack of

FoxP3 in humans leads to the fatal autoimmune disease,

IPEX. In addition, studies with the FoxP3-deficient mouse

model, also known as the scurfy mouse, have shown that

FoxP3 expression is profoundly important for the regulation

of the immune system and the development of Treg cells.

guava Cell Toxicity Kit Obtain all relevant statistics, including percentages of

target cells killed, effector and target cell percentages,

and viability data with this convenient kit. The assay uses

a well-characterized cell tracking dye that is optimized for

use on all guava easyCyte instruments. The dye diffuses

freely into cells and is retained within the cell without

affecting cellular function.

ORDERING INFORMATION Immunology Kits Description Qty/Pk Catalogue No.

FlowCellect Human FoxP3 Treg Characterization Kit

25 tests

FCIM025118

Flowcellect Human Lymphocyte ZAP-70 Characterization Kit • Anti-CD5 / Anti-CD19 / Anti-

ZAP-70

25 tests

FCIM025122

FlowCellect Mouse FoxP3 Treg Identification Kit• Anti-CD4 / Anti-FoxP3 clone 3G3

25 tests

FCIM025126

guava Cell Toxicity Reagents Kit• guava CFSE / 7-AAD

100 tests

4500-0230

FEATURED PRODUCTS

17

http://www.millipore.com/catalogue/item/FCIM025122&open&cid=BIOS-S-EPDF-1031-1105-DS







Advance your flow cytometry analysis with Millipore’s

growing selection of directly conjugated primary antibodies.

As a part of our complete benchtop flow cytometry solution

including instruments, software, service, and reagents,

Milli-Mark fluorescently-labeled antibodies are specifically

designed, optimized and validated for flow cytometry

applications.

Fig. 14. Fixed and permeabilized 2102Ep embryonal carcinoma cells were stained with Milli-Mark anti-hOct4-Alexa Fluor 488 (FCMAB113A4, green histogram) or IgG-Alexa Fluor 488 (grey histogram) at 1:100 for 1 hour and then analyzed by flow cytometry.

Fig. 15. Staurosporine-treated (yellow histogram) or untreated (grey histogram) Jurkat cells were stained using Milli-Mark pBcl2-(S70)-PE antibody (FCMAB140P) and analyzed using flow cytometry.

ORDERING INFORMATION Milli-Mark Stem Cell

Description Reactivity HostQty/Pk

Catalogue No.

Anti-hOCT4-Alexa Fluor 488

HumanMouse

Mouse 100 tests

FCMAB113A4

Anti-SSEA-4-PE Human Mouse 100 tests

FCMAB116P

Anti-Sox2-FITC HumanMouseRat

Mouse 100 tests

FCMAB112F

Anti-TRA160-FITC Human Mouse 100 tests

FCMAB115F

Anti-HESCA-1-FITC HumanMouse

Mouse 100 tests

FCMAB111F

Anti-SSEA-1-PE HumanMouseRat

Mouse 100 tests

FCMAB117P

Anti-SSEA3 Alexa 488, clone MC-631

Human Rat 100 tests

FCMAB141A4

Anti-Human Nuclei -FITC, clone 3E1.3

Human Mouse 100 tests

FCMAB157F

Anti-BMP-7-FITC, clone 2A10


FCMAB135F

Anti-mOCT4-Alexa Fluor 488, clone 7F9.2

HumanMouse

Mouse 100 tests

FCMAB124A4

Anti-TRA-1-81-FITC,clone TRA-1-81


FCMAB132F

Anti-TRA-2-49-FITC,clone TRA-2-49/6E


FCMAB133F

Anti-SRF-FITC, clone 1E1


FCMAB137F

ORDERING INFORMATION Milli-Mark Apoptosis and Cancer


Catalogue No.

Anti-phospho-Bcl-2 (Ser70)-PE, clone 69-10C-2-10C-18

Human Rabbit 100 tests

FCMAB140P

Rat-α-Caspase-2-FITC Human Rat 100 tests

FCMAB158F

For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”

Apoptosis & Cancer

Milli-Mark Conjugated Antibodies

18

http://www.millipore.com/flowcytometry/fc4/immunology&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/antibodies/ab/abhome&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/catalogue/item/FCMAB113A4&open&cid=BIOS-S-EPDF-1031-1105-DS


http://www.millipore.com/catalogue/item/FCMAB140P&open&cid=BIOS-S-EPDF-1031-1105-DS


http://www.millipore.com/catalogue/item/FCMAB158F&open&cid=BIOS-S-EPDF-1031-1105-DS













Fig. 17. HeLa cells were stained with either Milli-Mark anti-Syntaxin-3-FITC (FCMAB155F, green histogram) or IgG1 isotype control (grey histogram) and analyzed by flow cytometry.

Fig. 16. HeLa cells were stained with either Milli-Mark anti-Y14-FITC, clone 4C4 (FCMAB151F, green histogram) or with IgG2b isotype control antibody (grey histogram) and analyzed using flow cytometry.

ORDERING INFORMATION Milli-Mark Epigenetics and Gene Regulation


Catalogue No.

Anti-MAD2A-FITC, clone 17D10


FCMAB150F

Anti-Y14-FITC, clone 4C4


FCMAB151F

Anti-FXR1-FITC, clone 6BG10


FCMAB152F

Anti-SMN-FITC, clone 2B1


FCMAB153F

Anti-CAF1 p150-FITC, clone SS1, 1-3


FCMAB145F

Anti-TBX21/ T-Bet-FITC


FCABS131F

Anti-RPA2 p34 -FITC, clone RPA20 1-46


FCMAB143F

Anti-RPA1 p70 -FITC, clone RPA9, 1-30


FCMAB144F

Anti-BMI1-FITC, clone AF27


FCMAB149F

Anti-RBMS1-FITC,clone 4D11


FCMAB123F

Anti-c-Jun-FITC, clone 6E4

HumanMouseRat

Mouse 100 tests

FCMAB122F

Anti-RBMS1-FITC,clone 4D11

HumanMouse

Mouse 100 tests

FCMAB125F

Anti-BrdU-Alexa 488 Human Mouse 100 tests

FCMAB101A4

Phospho-ATM (Ser1981) - PE

HumanMouseRat

Mouse 100 tests

FCMAB110P

Phospho H3 (Ser10)- Alexa 488


FCMAB104A4

Phospho-SMC1 (Ser957) - Alexa 488

HumanBovineXenopus

Mouse 100 tests

FCMAB108A4

Anti-Cyclin B1 - PE HumanMouse

Mouse 100 tests

FCMAB102P

For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”

Epigenetics & Gene Regulation

ORDERING INFORMATION Milli-Mark Neuroscience


Catalogue No.

Anti-Syntaxin-3 FITC, clone 1-146


FCMAB155F

Anti-Rapsyn-FITC, clone 1234

Mouse Mouse 100 tests

FCMAB134F

Neuroscience

19











http://www.millipore.com/catalogue/item/FCMAS131F&open&cid=BIOS-S-EPDF-1031-1105-DS












Fig. 18. Flow cytometry analysis of human lymphocytes stained with Milli-Mark anti-CD4-FITC (FCMAB170F).

Fig. 19. A431 cells were stained with either Milli-Mark anti-EGFR-PerCP (FCMAB129CP, red histogram) or isotype control (grey histogram) and analyzed by flow cytometry.

ORDERING INFORMATION Milli-Mark Inflammation/Immunology


Catalogue No.

Anti-CD1a-FITC, clone NA1/34


FCMAB166F

Anti-CD2-FITC, clone MT910


FCMAB167F

Anti-CD3-PECy5, clone UCHT1


FCMAB169C5

Anti-CD4-FITC, clone MT310


FCMAB170F

Anti-CD8-FITC, clone DK25


FCMAB176F

Anti-CD11c-FITC, clone KB90


FCMAB179F

Anti-CD13-FITC, clone WM-47


FCMAB180F

Anti-CD14-FITC, clone TUK4


FCMAB181F

Anti-CD16-FITC, clone DJ130c


FCMAB183F

Anti-CD19-APC, clone HD37


FCMAB185AP

Anti-CD27-FITC, clone M-T271


FCMAB191F

Anti-CD45-PE, clone F10-89.4


FCMAB118P

Anti-CD45RA-FITC, clone MEM 56

HumanMouse

Mouse 100 tests

FCMAB126F

Anti-CD56-PE, clone MOC-1

Human Mouse Mouse

100 tests

FCMAB200P

Anti-CD57-FITC, clone TB01

Human Mouse Mouse

100 tests

FCMAB201F

ORDERING INFORMATION Milli-Mark Cell Signaling


Catalogue No.

Anti-EGFR-PerCP, clone LA22


FCMAB129CP

Anti-mTOR-FITC, clone 2ID8.2


FCMAB154F

Anti-Ras-FITC, clone RAS10


FCMAB148F

Anti-GβL/mLST8, clone 3E1.2, FITC Conjugate

HumanMouse

Mouse 100 tests

FCMAB121F

Anti-Akt/PKB-Alexa Fluor 647, clone SKB1


FCMAB128A6

Phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE

HumanMouseRat

Rabbit 100 tests

FCMAB100P

Anti-Ki-67-APC Human Mouse 100 tests

FCMAB103AP

Phospho-STAT1 (Y701) Alexa 488

HumanMouseRat

Mouse 100 tests

FCMAB106A4

Anti-β-catenin-PE, clone 7F7.2

Mouse Mouse 100 tests

FCMAB1209P

Phospho STAT5A/B (Y694/699) –PEH

HumanMouseBovineSheep

Mouse 100 tests

FCMAB105P

Inflammation/Immunology Cell Signaling

For a complete listing of Milli-Mark Antibodies visit: millipore.com/antibodies and search keyword, “Milli-Mark.”20



http://www.millipore.com/catalogue/item/FCMAB129CP&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/catalogue/item/FCMAB129CP&open&cid=BIOS-S-EPDF-1031-1105-DS




http://www.millipore.com/catalogue/item/FCMAB169C5&open&cid=BIOS-S-EPDF-1031-1105-DS












http://www.millipore.com/catalogue/item/FCMAB103AP&open&cid=BIOS-S-EPDF-1031-1105-DS




http://www.millipore.com/catalogue/item/FCMAB185AP&open&cid=BIOS-S-EPDF-1031-1105-DS





InstrumentsThe guava easyCyte flow cytometry systems are

uncomplicated instruments that deliver complex cell

analysis—right on your benchtop. The culmination of over

a decade of flow cytometry expertise, these instruments

use smaller sample, generate less waste, and are easier to

use and maintain than traditional flow cytometers—all while

providing the power you need in the most compact format

available. These advantages are made possible by our

patented microcapillary flow cell technology.

Microcapillary Flow Cytometry TechnologyAt the heart of every guava easyCyte system is a unique,

microcapillary flow cell that eliminates the need for sheath

fluid. This translates into smaller samples, less reagents

and minimal waste—saving you both time and money.

Because the flow cell is self-aligning and user-replaceable,

you can remove it yourself at any time for cleaning and

maintenance—no more expense or downtime for service

visits. And by eliminating complicated fluidics, we’ve created

a tiny instrument footprint that fits into the tightest spots,

saving valuable laboratory space.

Components Of The

Guava Flow Cytometry

Solution

Sheath Fluid: ~10 mL/testWaste: 8,000 mL per 8 hour runTypical #Cells Per Protocol: 100,000 - 1,000,000 cells/test

Sheath Fluid: NoneWaste: < 80 mL per 8 hour runTypical #Cells Per Protocol: 1,000 - 10,000 cells/test

Traditional Microcapillary

Waste

SampleSheath Fluid

Laser

Traditional sheath fluid system Guava-patented microcapillary system

Laser

She

ath

Flow

Inje

ctio

n Tip

Waste

Sam

ple

Flow

Sample

Laser

WasteLaser

Waste

Flow Cell

Sample

Sam

ple

Flow

21


Small footprint saves valuable laboratory space

Width: 20.3 in (51.5 cm)

Depth: 23.4 in (59 cm)

Height: 10.0 in (25.4 cm) (does not include laptop)

Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell

Waste vial collects less than 80 mL of waste in a typical 8-hour workday

Robotic sample tray provides walk-away automation for a 96-well microplate and up to 10 sample tubes

Microcapillary flow cell requires no sheath fluid and

is user-replaceable

Up to six-color detection made possible by one (blue) or

two excitation lasers (blue & red)

SPECIFICATIONS

System easyCyte 5HT easyCyte 6HT easyCyte 6HT-2L easyCyte 8HT

Catalogue # 0500-4005 0500-4005 0500-4007 0500-4008

Option # N/A 0500-4006 N/A N/A

Laser Blue (488 nm) Blue (488 nm)Blue (488 nm) and

Red (640 nm)Blue (488 nm) and

Red (640 nm)

FSC

SSC

Green 525/30 nm 525/30 nm 525/30 nm 525/30 nm

Yellow 583/26 nm 583/26 nm 583/26 nm 583/26 nm

Red1 680/30 nm 680/30 nm 690/50 nm 690/50 nm

NIR1 N/A 785/70 nm N/A 785/70 nm

Red2 N/A N/A 661/19 nm 661/19 nm

NIR2 N/A N/A N/A 785/70 nm

Microcapillary Fluidics

Direct, Absolute Cell Counts

Automation – 96 Well and 10 Tubes

Mixing

Dell® Latitude® E6500 Laptop with Intel® Core 2 Duo (P8600), 2.40 GHz, 1066 MHz FSB

InCyte™ Software

guavaSuite Software Modules

Digital Signal Processing

guava easyCyte High Thoughput Sampling Instruments

22






Small footprint saves valuable laboratory space

Width: 17.75 in (45.1 cm)

Depth: 17.25 in (44.5 cm)

Height: 8.75 in (22.2 cm) (does not include laptop)

Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell

Single sample loader Swivel arm functionality, holds two tubes and allows instant acquisition

Waste vial collects less than 80 mL of waste in a typical 8-hour workday

Up to six-color detection made possible by one (blue) or

two excitation lasers (blue and red)

Microcapillary flow cell requires no sheath fluid and is

user-replaceable

SPECIFICATIONS

System easyCyte 5 easyCyte 6 easyCyte 6-2L easyCyte 8

Catalogue # 0500-5005 0500-5005 0500-5007 0500-5008

Option # N/A 0500-5006 N/A N/A

Laser Blue (488 nm) Blue (488 nm)Blue (488 nm) and Red

(640 nm)Blue (488 nm) and Red

(640 nm)

FSC

SSC

Green 525/30 nm 525/30 nm 525/30 nm 525/30 nm

Yellow 583/26 nm 583/26 nm 583/26 nm 583/26 nm

Red1 680/30 nm 680/30 nm 690/50 nm 690/50 nm

NIR1 N/A 785/70 nm N/A 785/70 nm

Red2 N/A N/A 661/19 nm 661/19 nm

NIR2 N/A N/A N/A 785/70 nm

Microcapillary Fluidics

Direct, Absolute Cell Counts

Automation – 96 Well and 10 Tubes

N/A N/A N/A N/A

Mixing N/A N/A N/A N/A

Dell Latitude E6500 Laptop with Intel Core 2 Duo (P8600), 2.40 GHz, 1066 MHz FSB

InCyte Software

guavaSuite Software Modules

Digital Signal Processing

guava easyCyte Single Sample Instruments

23






24

ORDERING INFORMATION Flow Cytometry SystemsDescription Catalogue No.

Single Sampling Instruments

guava easyCyte 5 Base System 0500-5005

guava easyCyte 6-2L Base System 0500-5007

guava easyCyte 8 Base System 0500-5008

PCA Base System 0500-1090

High Throughput Sampling Instruments

guava easyCyte 5HT Base System 0500-4005

guava easyCyte 6HT-2L Base System 0500-4007

guava easyCyte 8HT Base System 0500-4008

PCA-96 Base System 0100-8710

SOFTWAREYour specific research needs are always changing, and

Millipore’s guava optimized software modules are flexible

enough to accommodate you at every level. Because all our

software modules use the same, intuitive user interface, it’s

easy to switch from one to another. The guavaSoft suite is

best for user-defined assays, the assay-specific software

modules can provide the most focused data output, and

the InCyte software package enables easy visualization

of data in a broad biological context. You can export to

spreadsheets or any third-party analysis format, and easily

attain 21 CFR part 11 compliance with all our software

packages.

Drag-and-drop gating from one plot to another

View up to 11 plots at once

Real-time plot adjustments

Organize acquired data sets and select individual wells for display

Quickly link to and review previously analyzed data

Easily cre-ate analysis templates

Combine groups of data and analysis templates to construct heat maps or EC

50 curves

Slider bars set cut-offs or threshold values for each experimental sector

Heat map shows values across an entire plate

InCyte: IntuitiveInCyte software is the first analysis package designed

specifically to give every user the power to draw

conclusions about the biological significance of data. It has

an intuitive, easy-to-use interface that makes it possible

to visualize and compare up to eight data sets at the same

time. The software is ideally suited for interrogation of high

content data sets derived from multiple functional studies.

InCyte software brings a new level of analytical power to

flow cytometry, enabling users to analyze entire plates of

data in less time than it usually takes to analyze a single

sample. In addition, data acquisition is fully incorporated

so that InCyte software can function as the primary data

acquisition and analysis package for the instrument. Most

importantly, comparative results are displayed at the

experiment level rather than on an individual well/sample

basis.









Start your benchtop cell analysis at: millipore.com/flowcytometry.

Protocols, products, webcast links, online demos and complete support are one click away.


Millipore, Advancing Life Science Together, guava, ViaCount are registered trademarks of Millipore Coporation.The M mark, Cell Cycle Stop, ChemiScreen, easyCyte, InCyte and Milli-Mark are trademarks of Millipore Corporation.Alexa Fluor is a registered trademark and MitoSOX is a trademark of Life Technologies, Inc.Dell and Latitude are registered trademarks of Dell, Inc. Intel is a registered trademark of Intel Corporation.Lit No. PB3945EN00, 09/10 LS SBU-10-03539Printed in U.S.A. and France© 2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

www.millipore.com/flowcapabilities

TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

In Europe, please call Customer Service:

France: 0825.045.645

Spain: 901.516.645 Option 1

Germany: 01805.045.645

Italy: 848.845.645

English UK: 0870.900.46.45

For other countries across Europe and the world,

please visit www.millipore.com/offices.

For Technical Service, please visit www.millipore.com/techservice.

http://www.millipore.com/flowcytometry/flx4/guavaform&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/?cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/offices/cp3/officeshome&open&cid=BIOS-S-EPDF-1031-1105-DS

http://www.millipore.com/techservices/techservices.do?open&cid=BIOS-S-EPDF-1031-1105-DS

Benchtop Flow Cytometry Solutions - Kits & Reagents

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